3H-Substance P(3H-SP), 3H-eledoisin (3H-ELE), and 125I-substance K (125I-SK) have been utilized to examine the characteristics of tachykinin binding sites in mammalian tissues. 3H-Sp, 3H-ELE, and 125I-SK binding in rat salivary gland membranes is inhibited by tachykinins in the rank order of SP Greater than or Equal to physalaemin Greater than SK Greater than ELE Greater than kassinin which is consistent with the existence of only a SP-P type receptor in this tissue. In smooth muscle membranes from guinea-pig small intestine and rat duodenum, 125I-SK binding is inhibited in the rank order of SK Greater than ELE Greater than kassinin Greater than SP Greater than or Equal to physalaemin which is distinct from SP-P type binding and from the SP-E type receptor suggested by in vitro bioassays. The gut appears to contain a unique SK binding site which may be the receptor for SK released from neurons in which the peptide coexists with SP. There is an asymmetric distribution 125I-SK binding sites throughout the rat gastrointestinal tract and the rat has substantially more of these sites in the pylorus, fundus, duodenum, and colon than does the guinea pig, 125I-SK also binds to sites in the rat brain. 3H-SP binds presumably to the SP-P receptor in rat brain and the levels of this binding are transiently elevated during the last prenatal and first postnatal week. 3H-SP also binds to high affinity sites in bovine adrenal cortex and medulla. We have been unable to demonstrate binding of 3H-SP, 3H-ELE, or 125I-SK to intact cultured cells from WKY and SHR rat aortae.